Difference between revisions of "IC4R010-Proteomic-2004-14730683"
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==Plant Culture & Treatment== | ==Plant Culture & Treatment== | ||
==Protein Extraction and 2-D PAGE== | ==Protein Extraction and 2-D PAGE== | ||
| + | *Aliquots (1 g) of synchronized cell samples were ground to a fine powder in liquid nitrogen. Protein extraction and | ||
| + | precipitation was performed in 10% v/v TCA in acetone with 0.07% v/v b-mercaptoethanol at 2207C for 1 h, followed by centrifugation for 10 min at 47C, 50 0006g. The pellets were washed twice with cold acetone containing 0.07% b-mercaptoethanol and centrifuged for 10 min at 47C, 50 0006g, and the pellets dried. Proteins were solubilized in 500 mL of lysis buffer (9 M urea, 4% w/v CHAPS,0.5% v/v Triton X-100, 0.5% IPG buffer, 3 mM tributylphosphine (TBP)). Finally, the sample was centrifuged 10 min at 207C, 60 0006g. Concentration of the supernatant was determined using protein assay (2D-Quant kit; Amersham, UK). | ||
| + | <br> | ||
==Research Findings== | ==Research Findings== | ||
Revision as of 10:19, 17 July 2016
Contents
Project Title
- Proteome analysis of cultivar-specific deregulations of Oryza sativa indica and O. sativa japonica cellular suspensions undergoing Rice yellow mottle virus infection
The Background of This Project
Plant Culture & Treatment
Protein Extraction and 2-D PAGE
- Aliquots (1 g) of synchronized cell samples were ground to a fine powder in liquid nitrogen. Protein extraction and
precipitation was performed in 10% v/v TCA in acetone with 0.07% v/v b-mercaptoethanol at 2207C for 1 h, followed by centrifugation for 10 min at 47C, 50 0006g. The pellets were washed twice with cold acetone containing 0.07% b-mercaptoethanol and centrifuged for 10 min at 47C, 50 0006g, and the pellets dried. Proteins were solubilized in 500 mL of lysis buffer (9 M urea, 4% w/v CHAPS,0.5% v/v Triton X-100, 0.5% IPG buffer, 3 mM tributylphosphine (TBP)). Finally, the sample was centrifuged 10 min at 207C, 60 0006g. Concentration of the supernatant was determined using protein assay (2D-Quant kit; Amersham, UK).
